Gastric Ulceration in Horses the Role of Bacteria and Lactic Acid

8/3/2019 Gastric Ulceration in Horses the Role of Bacteria and Lactic Acid

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Gastric Ulceration in Horses

The role of bacteria and lactic acid

RIRDC Publication No. 08/033

RIRDCInnovation for rural Australia

HORSES


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Gastric Ulceration in Horses

The role of bacteria and lactic acid

By Dr Rafat Al Jassim, Dr Thomas McGowan, Prof Frank Andrews and Dr Catherine McGowan

October 2008

RIRDC Publication No 08/033RIRDC Project No UQ-115A


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2008 Rural Industries Research and Development Corporation.All rights reserved.

ISBN 1 74151 622 6ISSN 1440-6845

Gastric Ulceration in Horses: The role of bacteria and lactic acid

Publication No. 08/033Project No. UQ-115A

The information contained in this publication is intended for general use to assist public knowledge and discussionand to help improve the development of sustainable regions. You must not rely on any information contained inthis publication without taking specialist advice relevant to your particular circumstances.

While reasonable care has been taken in preparing this publication to ensure that information is true and correct,the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.

The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), theauthors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability toany person, arising directly or indirectly from any act or omission, or for any consequences of any such act or

omission, made in reliance on the contents of this publication, whether or not caused by any negligence on thepart of the Commonwealth of Australia, RIRDC, the authors or contributors.

The Commonwealth of Australia does not necessarily endorse the views in this publication.

This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights arereserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rightsshould be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact DetailsDr Rafat Al Jassimc/o School of Animal StudiesThe University of Queensland

Gatton 4343

Phone: 07 5460 1521Fax: 07 5460 1444Email: [email protected] submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact DetailsRural Industries Research and Development CorporationLevel 2, 15 National CircuitBARTON ACT 2600PO Box 4776KINGSTON ACT 2604

Phone: 02 6271 4100Fax: 02 6271 4199Email: [email protected]: http://www.rirdc.gov.au

Published in October 2008 by Union Offset

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ForewordGastric ulceration is widespread and a very common problem in horses in training. When it occurs in

horses, gastric ulceration is a potential insidious cause of poor athletic performance or, when severe,

an animal welfare concern. It is widely accepted that this is a problem resulting from feeding and

management practices, especially in racehorses where the prevalence is extremely high. Racehorses

are fed large meals of grain rich diets and with extended periods of fasting between meals. Thiscombined with increased gastric acid production during exercise, reduction in saliva production due to

a low fibre diet, and indoor confinement, is likely to contribute to the development of stomach ulcers.

Recent work has indicated the presence of diverse microbial populations that survive the stomach

environment of both starved and fed horses. Recent work in the USA has shown that volatile fatty

acids produced by bacteria in the stomach of horses were associated with increased ulcer severity

(Nadeau et al., 2000) and have a potent in vitro effect on reducing mucosal integrity, an effect even

greater than that by normal gastric acid. A theory was developed: bacteria and their products,

especially lactic acid and volatile fatty acids by bacteria in the non-glandular region of the stomach,

play a vital role in the development and progression of gastric ulcers in horses. Identification of the

key acid producing bacteria and dietary regimens that minimise their multiplication will aid in the

development of strategies to control gastric ulcers in horses. This will undoubtedly help in reducingcost of medical treatment of gastric ulcers in racehorses and improve their health, welfare and

performance.

The results of this project have confirmed the diverse population of bacteria that live within the normal

equine stomach. Importantly, it was found that the diversity of bacteria adherent to the stomach lining

decreases during ulceration, which implies the potential development of a dominant population of

pathogenic organisms. Lactic acid, produced in the stomach of horses increases the permeability of the

equine stomach and this may be important in the pathogenesis of gastric ulcers (worsening or causing

gastric ulceration). The project went further to both develop a model of dietary induced gastric

ulceration and monitor natural recovery at pasture and with treatments aimed at microbial populations.

The results indicated that gastric ulceration could be induced in stabled horses rapidly by increasing

the concentrate grain based portion of the ration to 5 kilograms per day, and that the severity worsened

when the roughage was restricted to three kilograms per day, similar to what is common practice

amongst racehorse trainers in Australia. Lastly, treatment of gastric ulceration with oral antibiotics

decreased the severity of gastric ulceration. There was a trend for the same effect with administration

of a live bacterial culture probiotic as a treatment.

In conclusion, the role of bacteria in the pathogenesis of gastric ulceration in horses has been

established through laboratory experiments and studying gastric ulceration in the live horse. A role for

modification of microbial populations in the future treatment of gastric ulceration has been shown and

the future use of probiotics and other non-medical manipulations of microbial populations in horse

predisposed to gastric ulceration is a promising prospect.

This project was funded from industry revenue that is matched by funds provided by the Australian

Government. This report, an addition to RIRDCs diverse range of over 1800 research publications,forms part of our Horses R&D program, which aims to assist in developing the Australian horse

industry and enhancing its export potential.

Most of our publications are available for viewing, downloading or purchasing online through our

website:

downloads at www.rirdc.gov.au/fullreports/index.html

purchases at www.rirdc.gov.au/eshop

Peter OBrien

Managing Director

Rural Industries Research and Development Corporation

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Acknowledgments

The authors would like to acknowledge the help of our research assistants Lillian Jedski and

Kelly Jamieson for their organisation, excellent care and love for the horses and interest in the project.

Abbreviations

ADF acid detergent fibre

Ash mineral ash

CP crude protein

DE digestible energy

DGGE denaturing gradient gel electrophoresis

DM dry matter

G conductance

HCl hydrochloric acidH2 histamine type 2

Isc short-circuit current

LA lactate

LAB lactic acid producing bacteria

LRS lactated Ringers solution

mM millimolar

NDF neutral detergent fibre

NG non-glandular mucosa of the stomach

NRS in normal Ringers solution

OM organic matter

PD potential difference

R electrical resistance

TB Thoroughbred

VFA volatile fatty acids

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Contents

Foreword ............................................................................................................................................... iii

Acknowledgments................................................................................................................................. iv

Executive Summary ............................................................................................................................viiIntroduction ........................................................................................................................................... 1

Animal welfare implications of gastric ulceration .............................................................................. 1The stomach of the horse..................................................................................................................... 1Microbiology of the stomach of the horse........................................................................................... 2Pharmaceutical treatment of gastric ulcers.......................................................................................... 3

Objectives............................................................................................................................................... 4

Methodology .......................................................................................................................................... 5Study 1. The bacterial community of the horse stomach

,.................................................................... 5

Protocol ............................................................................................................................................... 5Study 2: In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric

squamous mucosa................................................................................................................................ 6Specific Objectives.............................................................................................................................. 6Techniques used .................................................................................................................................. 6Analytical methods.............................................................................................................................. 7Study 3: Induction and recovery of dietary induced gastric ulcers in horses...................................... 7Protocol ............................................................................................................................................... 7Study 4: Treatment of dietary induced gastric ulcers in horses......................................................... 10

Results .................................................................................................................................................. 12Study 1. The bacterial community of the horse stomach .................................................................. 12Study 2: In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric

squamous mucosa.............................................................................................................................. 14

Study 3: Induction and recovery of dietary induced gastric ulcers in horses.................................... 15Study 4: Treatment of dietary induced gastric ulcers in horses......................................................... 18

Analysis of the Severity of gastric ulceration................................................................................ 19Analysis of the Number of gastric ulceration................................................................................ 19

Discussion of results ............................................................................................................................ 20Bacterial Community of the equine stomach .................................................................................... 20The role of lactic acid........................................................................................................................ 20Induction of gastric ulcers ................................................................................................................. 21Treatment of gastric ulcers ................................................................................................................ 21

Implications.......................................................................................................................................... 22

Recommendations ............................................................................................................................... 23

References ............................................................................................................................................ 24Appendices ........................................................................................................................................... 26

List of presentations .......................................................................................................................... 26Publications ....................................................................................................................................... 26


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List of Figures

Figure 1. Gastroscopy examination procedure. From the left: Dr Rafat Al Jassim, Dr Thomas

McGowan, Horse 4, and Dr Catherine McGowan

Figure 2. Phylogenetic relationship of the derived sequences from 16S rDNA of cultured bacteria

Figure 3. Clones derived from denaturing gradient gel electrophoresis (DGGE) bands identified using

the V3 region of 16S rDNA. Genomic DNA was extracted from stomach contents and stomach

mucosa

Figure 4. This graph represents the mean body weight for each week of trial 1 (95% confidence

interval error bars)

Figure 5. mean gastric ulcer score for 13 horses fed concentrate ration and being confined during

weeks 0 10 and subsequently during pasture recovery during weeks 10-16. Number and severity are

shown separately as blue and purple respectively

Figure 6. Endoscopic images of severity grade 3 ulceration in 2 horses.

Figure 7. Graph representing the mean number scores for the 3 different groups in the study (Control,

Pro-biotic, and Antibiotic) for each week during trial 2 (95% CI error bars)

Figure 8. Graph representing the mean severity score for each of the 3 groups (Control, Probiotic and

Antibiotic) for each week during the study

List of TablesTable 1. Chemical composition of the hay and concentrate mixture fed to horses

Table 2. composition of the concentrate mix

Table 3. Grading system for gastric ulcers based on MacAllister et al., 1997

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Executive Summary

What the report is aboutThis report is about the role that bacteria and lactic acid plays in the development of gastric ulceration

in horses. It also explores the use of contrasting diets to determine whether they contribute to the build

up of lactic acid and volatile fatty acids. Identification of the key acid producing bacteria and dietaryregimens that minimise their multiplication will aid in the development of strategies to control gastric

ulcers in horses. This will undoubtedly help in reducing the cost of gastric ulcer treatment in

racehorses and improve their health and performance.

BackgroundGastric ulceration is widespread and a very common problem in horses in training. It is widely

accepted that this is a problem resulting from feeding and management practices. Racehorses are fed

large meals of grain rich diets and usually fasted for an extended period before exercise. It is the

combination of increased gastric acid production during exercise, reduction in saliva production due to

a low fibre diet, and indoor confinement that likely contributes to the development of stomach ulcers.

Physiologically, it is claimed that sudden increases in blood sugar supply elevates gastrin production,

which in turn increases gastric acid production. This leads to prolonged periods of exposure of theunprotected region of the stomach to acid, which is a major cause of ulceration.

Recent work in the US has shown that volatile fatty acids (VFAs) produced in the stomach of horses

are associated with increased ulcer severity (Nadeau et al., 2000) and have a potent in vitro effect on

reducing mucosal integrity, an effect even greater than that by normal gastric acid (HCl). This

reinforces the concept that bacterial fermentation products play a role in the pathogenesis and

persistence of gastric ulceration in horses (Nadeau et al., 2003a and b). An early report on the

microbiology of fermentative acidosis in grain fed animals by Al Jassim and Rowe (1999) showed that

Streptococcus bovis and Streptococcus equinus are the key lactic acid producing bacteria of the equine

gastrointestinal tract. Recent work in our laboratory indicated the presence of a diverse microbial

population that survive in the stomach environment of both starved and fed horses.

We therefore developed a theory that bacteria and their products, especially lactic acid and VFAs by

bacteria in the non-glandular region of the stomach, are vital in the development and progression of

gastric ulcers in horses.

AimThe major aims of the project were:

1. To establish involvement of lactic acid producing bacteria and their products (VFA & lactic acid) in

the pathogenesis of gastric ulceration in horses.

2. To determine the effect of contrasting dietary regimens on the microbial contribution to the build-up

of lactic acid and VFAs in the non-glandular portion of the stomach.

ObjectivesIn order to address these aims, we divided the research into four studies with the following objectives:

Study 1: to determine the bacterial community of the normal horse stomach and compare that with the

bacterial community of the ulcerated stomach

Study 2: to determine the in vitro effects of hydrochloric and lactic acids on bioelectric properties of

equine gastric squamous mucosa

Study 3: To develop a model for induction and recovery of dietary induced gastric ulcers in horses

Study 4: To investigate treatment of dietary induced gastric ulcers in horses using modification of

bacterial populations.

Key findings

The results of this project have shown that there is a diverse population of bacteria that live within thenormal equine stomach, but the diversity of bacteria adherent to the stomach epithelium decreases

during ulceration that implies the potential development of a population of pathogenic organisms.

Lactic acid, produced in the stomach of horses increases the permeability of the equine stomach (non-

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glandular mucosal), and this may be important in the pathogenesis of gastric ulcers. The project went

further to develop a model of dietary induced gastric ulceration, to monitor natural recovery on pasture

and to use treatments aimed at microbial populations. The results indicated that we could induce

gastric ulceration in stabled horses rapidly by increasing the concentrate grain based portion of the

ration to five kilograms per day. Also the severity worsened when the roughage was restricted to three

kilograms per day; similar to what is common practice amongst racehorse trainers in Australia. Lastly,

treatment of gastric ulceration with oral antibiotics decreased the severity of gastric ulceration. Therewas a trend for the same effect with administration of a live bacterial culture probiotic as a treatment.

ImplicationsThe implications of this research are that we have established the role of bacteria in the pathogenesis

of gastric ulceration in horses through laboratory experiments and studying gastric ulceration in the

live horse. There is a great opportunity to further investigate the use of antibiotics/and or probiotics for

treatment of ulcers in horses. A role for modification of microbial populations in the future treatment

of gastric ulceration has been shown and the future use of probiotics and other non-medical

manipulations of microbial populations in horse predisposed to gastric ulceration is a promising

prospect.

RecommendationsThe role of bacteria in the pathogenesis of gastric ulceration in horses has been established through

laboratory experiments and studying gastric ulceration in the live horse. A role for modification of

microbial populations in the future treatment of gastric ulceration has been shown and the future use of

probiotics and other non-medical manipulations of microbial populations in horse predisposed to

gastric ulceration is a promising prospect. It is recommended that further studies on the use of different

live bacterial probiotics on the prevalence of gastric ulceration. It is also recommended that restriction

of roughage in horses on a high grain diet should be avoided and that horse owners feeding their

horses five kilograms of high grain based concentrate diet per day should be aware of the very high

prevalence of gastric ulceration under this regimen, especially if the horse is concurrently confined to a

stable.

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Introduction

Gastric ulceration is a widespread and very common problem in horses in training. Thoroughbred

racehorses usually have the highest incidence of stomach ulceration, affecting more than 80% of

horses in training (Vatistas et al., 1994). It is widely accepted that this is a problem resulting from

feeding and management practices. Racehorses are fed twice daily of grain rich diets and usuallyfasted for an extended period before exercise. It is the combination of increased gastric acid production

during exercise, reduction in saliva production due to low fibre content of the diet, and indoor

confinement that likely contributes to the development of stomach ulcers. Physiologically, it is

claimed that sudden increases in blood sugar supply elevates gastrin production, which in turn

increases gastric acid production. This will lead to prolonged periods of exposure of the unprotected

region of the stomach to acid, which is a major cause of ulceration. Gastric ulcers in the non-glandular

region of the stomach are likely caused by exposure to organic acids (hydrochloric [HCl], volatile fatty

[VFAs], and bile acids) and the inadequate barrier defences, including the lack of a thick mucus layer

and bicarbonate (Nadeau et al., 2000; Berschneider et al., 1999; Bullimore et al., 2001; Nadeau et al.,

2003a; Nadeau et al., 2003b).

Horses with gastric ulceration may suffer weight loss and poor performance; some severe enough toresult in retirement from racing. This clearly has significant economic and welfare implications and

has serious cost implications to the equine industry. The exact economic impact of gastric ulcer

disease in horses is not exactly known, however, gastric ulcer prevalence estimates range from 25 to

81%, of which approximately 50% of horses have clinical signs of poor performance, colic, and

weight loss (Hammond et al., 1986). Furthermore, in a California study of racehorses gastric ulcer

severity was correlated with poor performance (Vatistas et al., 1994). This cost is not only in lost days

to training and racing as a result of poor performance or ill health, but also in lost days racing due to

withholding periods of medications currently used to treat gastric ulceration. This may result in

millions of dollars of lost revenue each year in training and racing days, and in the cost of treatment.

Furthermore, in severe cases, gastric ulcer disease can result in acute death due to fatal haemorrhage

and gastric rupture (Traub-Dagartz et al., 1985; Todhunter et al., 1986). If more were known about the

dietary factors involved in gastric ulcer disease pathogenesis, perhaps feeding practices could be

altered to decrease the incidence and prevalence of gastric ulcer disease in horses. Therefore, by

decreasing the incidence and prevalence of gastric ulcer disease in horses, horse suffering and the

economic loss to the racehorse industry could be minimized.

Animal welfare implications of gastric ulcerationThe horse welfare issue has always been a key theme in RIRDC research programs. Previous programs

promoted knowledge about effective nutritional and housing management and emphasised the needs to

improve the health and welfare of the horse. As mentioned earlier, stomach ulceration is a man made

problem often associated with concentrate feeding, stabling, intensive exercise and transport.

Therefore, knowledge of the exact cause, will lead to the development of preventive measures, which

undoubtedly improve the welfare of the horse.

The stomach of the horseHorses are herbivores evolved to digest fibre. The digestive processes begin with enzymatic digestion

in the stomach and small intestines followed by extensive fermentation in the caecal and colon. The

stomach of the horse is relatively small comprising only 8-10% of the gastro-intestinal tract with a net

capacity of 7.5 to 15 litters, depending on feed type. The stomach has two distinct regions, the upper

half of the stomach consists of squamous epithelial cells that lack an apparent mucus layer, while the

lower part is glandular gastric with secretory tissue that has a mucous membrane lining.

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Horses on pasture graze continuously and maintain a full stomach. Food entering the stomach is

poorly mixed allowing a pH gradient between the entrance of the stomach, the cardiac region and the

pyloric, glandular region. Under normal roughage feeding conditions saliva is continuously delivered

to the stomach providing buffering to the region close to the entrance. The pH at the non-glandular

region is around 5.4 while that of the pyloric region is around 1.8. As a result, horses maintained

solely on pasture rarely develop digestive problems such as gastric ulcers. However, feeding grain or

concentrate mixes rich in energy and feed deprivation for prolonged periods increases the exposure ofthe stratified squamous epithelial mucosa of the non-glandular region, resulting in the development of

gastric ulcers (Murray and Eichorn, 1996).

Microbiology of the stomach of the horseAlthough the organism has been detected in the equine stomach (Contreras et al., 2007), there is no

evidence to suggest equine gastric ulcers are caused byHelicobacter pylori which is the bacterium that

is the common cause of ulcers in humans (Pagan, 1997). In fact, the microbial community of the

equine gastro-intestinal tract has received very little attention despite its importance to the health of the

animal. Available information has dealt mainly with the fermentation processes in the hindgut,

especially these related to fibre digestion (Lin and Stahl, 1995; Julliand et al., 1999; Daly et al., 2001)

and fermentative acidosis (Al Jassim and Rowe, 1999).

However, recent developments and use of molecular techniques have shown the bacterial diversity

within the equine large intestine (Daly et al., 2001) and to a lesser extent that of the stomach (Scott

et al., 2003). Bacterial fermentation starts in the stomach despite the mild acidic conditions of the non-

glandular region of the stomach, with the production of VFA and lactic acid. Fermentation may cease

at the more acidic glandular region of the stomach but lactic acid producing bacteria remain viable

when horses are deprived of food for a period longer than 12 hours and seem to tolerate acid shock.

An early report on the microbiology of fermentative acidosis in grain fed animals by Al Jassim and

Rowe (1999) showed that Streptococcus bovis and Streptococcus equinus are the key lactic acid

producing bacteria of the equine gastrointestinal tract. Previous work in our laboratory indicated the

presence of a diverse microbial population that could survive the stomach environment of both starvedand fed horses. More recently 25 bacterial isolates were selected on the basis of their dominance and

lactate production from different parts of the gastrointestinal tract of healthy and laminitis induced

horses. Sequence analysis of their 16S rDNA indicated that most of the isolates were very closely

related to species of the genusLactobacillus, includingLact. mucosae,Lact. delbrueckii, and

Lact. salivarius (Al Jassim et al., 2005). Interestingly, some isolates were very closely related to

Mitsuokella jalaludinii, a strong D-lactate producing bacterium. Little is known about other members

of the lactic acid producing bacteria of the equine stomach and gastrointestinal tract.

Aggressive lactic acid producing bacteria in the equine stomach have recently been identified

produce L and D-lactate which has previously been undiscovered/not identified in the horse GIT.

Further, evidence has indicated build up of acids in the non-glandular region of the stomach due to

microbial fermentation of soluble carbohydrates leading to the production and accumulation of lacticacid. Recent work in our laboratories at Gatton showed that the concentration of lactic acids in the

non-glandular region of the stomach of horses fed grass and grains (4 kg grass hay and 4 kg dry-rolled

or steam-flaked sorghum) was higher than 40 mmol/l at 2 -6 h after feeding (Al Jassim 2006). The

same study showed that all the lactic acid is absorbed from the gastrointestinal tract before the contents

reaches the caecum. Absorption of lactic acid into the mucosa may cause some damage. As acid build

up is suspected to be the main cause for stomach ulcers in horses, it is therefore important to identify

and quantify the different contributors to acid build up.

Recent knowledge has provided new insight into a bacterial involvement in gastric ulceration. Recent

work in the US has shown that VFAs produced in the stomach of horses were associated with

increased ulcer severity (Nadeau et al., 2000). These acids have a potent in vitro effect on reducingmucosal integrity, an effect even greater than that by normal gastric acid (HCl) reinforcing the concept

that bacterial fermentation products play a role in the pathogenesis and persistence of gastric ulceration

in horses (Nadeau et al., 2003a and b).

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The hypothesis is that the production of acids, especially lactic acid, by these bacteria in the non-

glandular region of the stomach is the vital pre-disposing cause for the development of gastric ulcers.

Identification of the key lactic acid producing bacteria will aid the development of strategies to control

gastric ulceration. This will undoubtedly help in reducing costs of maintaining racehorses and improve

their health, welfare and performance.

Pharmaceutical treatment of gastric ulcersWhile there are treatments available for gastric ulcers, they are expensive; unable to be used close to

racing and do not target the specific pathogenesis nor prevent the development of gastric ulceration.

Recent knowledge has provided new insight into a bacterial involvement in gastric ulceration.

The use of ranitidine, a histamine type-2 receptor antagonist drug (Murray and Eichorn 1996), and

omeprazole an acid pump inhibitor (Andrews et al., 1999) have proved to work well to treat stomach

ulcers in horses. These drugs reduce the secretion of acid, but do not target microorganisms that

colonise the lesions or those responsible for the build up of acid in the non-glandular part of the

stomach. The cost of treatment is expensive and there is a high recurrence rate when treatment is

withdrawn, thus dietary management in horses would reduce costs and decrease suffering in horses.

Besides, horses continue to have ulcers even though they are maintained on acid suppressive doses of

medications. A more comprehensive approach to ulcer management is surely indicated to reduce the

cost of gastric ulcer disease in horses. The role of VFAs in causing acid injury may explain why

current gastric acid secretory inhibiting agents are not completely effective in healing gastric ulcers in

horses. For example, treatment with histamine type 2 (H2) receptor antagonists in 55 horses with

gastric ulceration lead to endoscopically confirmed resolution of lesions or improvement in only 32

horses. Of 32 horses treated with ranitidine for gastric lesions, there was significant improvement in

gastric lesion scores and in only 16 horses, complete healing. Omeprazole (0.7 mg/kg) given once

daily via nasogastric tube to 8 horses with chronic gastric cannulae was found to inhibit basal and

stimulated gastric acid output by 69% and 76% respectively and increased pH from 3.2 to 4.6 by the

5th day of treatment in basal gastric juice. In that same study, stimulated gastric juice pH increased

from 1.7 to 4.6 by the 5th day of treatment. Gastric ulcers were healed in only 8 of 12 horses by 18days and in 11 of 12 horses by 21 days of omeprazole treatment. Identification of the bacteria involved

in the pathogenesis of gastric ulceration will provide the necessary information for the development

and or selection of antibiotics for treatment of clinical cases.

Therefore, we proposed that bacteria are involved in the pathogenesis and or progression of equine

gastric ulcer signs and so specific treatment and prevention strategies can be developed to reduce the

incidence of gastric ulcers in horses. The aims of this project were then:

1: To establish involvement of bacteria and their products (VFAs and lactic acid) in the pathogenesis

of gastric ulceration in horses.

2: To determine the effect of contrasting dietary regimes on the microbial contribution to the build-upof lactic acid and VFAs in the non-glandular portion of the stomach.

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Methodology

Studies were approved by the Institutional Animal Ethics Committee.

Study 1. The bacterial community of the horse stomach1,2

This study involved the investigation of normal bacterial populations in the normal and abnormal

equine stomach using material obtained from an abattoir.

Protocol

Samples of stomach contents and lining were obtained from twenty horses post mortem. Stomach

contents were pooled into four groups of five horses each and transported immediately to the

laboratory for processing. One group consisted of samples from five horses with ulcerated stomachs.

In addition, mucosa from the glandular region and squamous tissue from the non-glandular region of

the stomachs were taken from healthy and ulcerated horses for identification of bacteria associated

with these tissues. Epithelial samples were washed with phosphate buffered saline (PBS) immediatelyafter sampling, placed in a sterile container and kept on ice during transport to the laboratory.

All samples were processed by:

1. Culturing and isolation of lactic acid producing bacteria.

2. Extraction of genomic DNA and analysis of bacterial diversity by denaturing gradient gel

electrophoresis (DGGE).

1. Culture, isolation and identification of lactate-producing bacteria

Stomach contents were cultured for enumeration of lactate-producing bacteria (bacteria that grew in

MRS agar medium). Samples were processed under CO2 and serially diluted in tenfold increments up

to a dilution of 1:107

. Then, three dilutions (1:105

, 1:106

and 1:107

) were used to inoculate roll tubescontaining MRS agar medium. A modified non-selective MRS roll tube medium, Oxoid, England (De

Man et al., 1960) was used as described by Al Jassim et al., (2003). After incubation for 48 h at 39 C,

counts were carried out. Then 45 colonies were picked from the roll tubes, transferred into BM10

broth medium (Caldwell and Bryant, 1966) supplemented with glucose (0.3% w/v) and cultured in roll

tubes again. The procedure was repeated twice. After another 48 h of incubation, the remaining

cultures were examined under a microscope for purity, morphology and Gram staining.

Genomic DNA was obtained from each of the pure isolates and the 16S rDNA was amplified by PCR.

The diversity of pure isolates was initially determined by restriction fragment length polymorphism

(RFLP) analysis, and sixteen selected isolates from each RFLP group were cloned and sequenced. A

more definitive analysis of population diversity was undertaken by comparing the near-complete

sequences of 16S rDNA with those found in public databases.

2. Denaturing gradient gel electrophoresis (DGGE)

At the laboratory, the epithelial samples were placed in bag containing 10 mL of PBS and

homogenized in a stomacher (Stomacher 400 Circulator, Seward Ltd., Thetford, UK) for two cycles of

30 s at 230 rpm. The samples were then strained through two layers of sterilized cheesecloth and the

filtrate was retained for DNA extraction. The filtrate was centrifuged at 13,000 rpm for five min at

1 R.A.M. Al Jassim, S. Denman, J.D. Hernandez, C. M. McGowan, F. M. Andrews, C. S. McSweeny The

bacterial community of the horse stomach. RRI INRA Gut Microbiology: 5th Biennial Meeting: Research to

Improve Health, Immune Response and Nutrition. Reprod. Nutr. Dev. 46 (2006) S7 P2 INRA, EDP Sciences,

2006.2 R.A.M. Al Jassim, C.M. McGowan and F.M. Andrews. Bacterial diversity and role of lactic acid in the

pathogenesis of acid injury in the non-glandular region of the equine stomach. Recent Advances in Animal

Nutrition in Australia 2007.

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4C. The pellet was then resuspended in 2 mL sterilized distilled water and vortexed. Genomic DNA

was extracted from all samples (gut lining and stomach contents) using centrifugation, lysis buffer and

bead beating. PCR was performed to amplify the V3 region of the 16S rDNA in preparation for

denaturing gradient gel electrophoresis (DGGE) using primers 341F + GC clamp and 518R (Muyzer

et al., 1993). Samples were then run under DGGE conditions for 18 h, after which the bands were

visualized using silver staining. Twenty-five bands were chosen for recovery of DNA for further

analysis. Plugs were taken from these bands for DNA analysis and sequencing. The DNA of the gelplugs was amplified by PCR using the primers previously described for amplification of the extracted

DNA prior to DGGE analysis and then subjected to electrophoresis using a 2% agarose gel. Purified

PCR products were then ligated into a pGEM-T Easy vector (Promega, Madison, USA) and

transformed into E. coli Top10 cells. Sequencing was performed on five transformed colonies using

the T7 primer found within the pGEMT vector.

Sequence data analysis

DNA sequence data was edited using Bioedit software (Hall, 1999) and characterization for the most

closely related sequence was performed by pairwise BLAST (Tatusova and Madden, 1999). The

contiguous sequences were aligned in a 16S rDNA database using the ARB software package

(http://www.arb-home.de/) and the phylogenetic tree was generated using the neighbourhood joining

methods of the ARB software package. Bootstrap analysis using 2000 replicates was performed usingPaup*4.0b 10 to ascertain the robustness of the tree topology.

Study 2: In vitro effects of hydrochloric and lactic acids onbioelectric properties of equine gastric squamous mucosa3

This study involved investigating the effect of lactic acid on the integrity of the lining of the horse

stomach using an Ussing chamber. This work was carried out by our collaborator Professor Frank

Andrews, at the University of Tennessee USA. The objectives of this study were to compare acute

tissue injury induced by different concentrations of LA at different pH (1.5, 4.0, or 7.0). In vitro

electrophysiological measurements will be correlated to histological evidence of cellular injury.

Specific Objectives

1. To expose the horses non-glandular stomach mucosa to varying concentrations of LA (0

[control], 5, 10, 20, 40 mmol) at different pH (1.5, 4.0, 7.0), using an in vitro Ussing chamber system

and measure potential difference (PD) across the tissue and short circuit current (Isc) to determine cell

viability and function.

2. To examine the non-glandular stomach mucosa in objective 1 using light microscopy to

determine area of histopathologic changes, so that these changes can be correlated with alterations in

PD across the tissue and Isc.

3. Mucosa from the non-glandular stomach of 15 horses not suffering from gastric disease wasobtained immediately after euthanasia. Information regarding sex, breed, and age of animal, recent

medical history and dietary history has been obtained.

4. Mucosal tissues were studied in the in vitro Ussing chamber system, exposed to the various

concentrations of LA at a pH of 1.5, 4.0, or 7.0.

5. PD across the tissue and Isc were recorded every 15 minute for each of the treatment

combination.

6. The tissue were examined under light microscopy for area of histopathologic changes using an

image analyzer and these changes were correlated to electrical values, LA concentration and pH.

Techniques used

3 F.M. Andrews, B.R. Buchanan, S.B. Elliott, R.A.M. Al Jassim, C.M. McGowan, and A.M. Saxton. In vitro

effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa. Accepted

EVJ special colic issue January 2008.

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Fresh gastric non-glandular mucosa from horses was studied using the in vitro Ussing chamber

system. Once the tissues were mounted in the Ussing chamber system, spontaneous PD and Isc were

measured using Ringers-agar bridges, whose composition was identical to the serosal solution bathing

the surface. Tissue resistance was calculated from the open circuit PD and from the current necessary

to nullify the PD, the Isc. The tissue was clamped at 100uA of current when the open circuit PD is < 1

mV, and the resulting PD was recoded.

The mucosal surface of the stomach tissue was perfused either with Ringers solution (control) or

Ringers solution with differing concentrations of LA added. The resulting PD and Isc were measured

for each LA concentration and pH. Once measurements are completed, the tissues were removed from

the chambers, and placed in 10% formalin. Formalin-fixed sections were embedded in paraffin,

sectioned at a thickness of 5 um, and stained with H&E for examination by light microscopy.

Analytical methods

Means and pooled SEM were calculated. Data were analyzed using the mixed procedure of SAS. The

model used for each tissue type and variable was a split-plot ANOVA with treatment (buffer

conditions) in the main plot and time and time X treatment interaction in the subplot. In the presence

of a significant time X treatment interaction, an ANOVA was performed at each point of test for

differences between or among means. A Dunnetts one-tailed test was used to determine whether any

mean value differs from the control value. Variables were correlated with area of histopathologic

change using a Pearson moment correlation coefficient. A P 0.05 was considered significant.

Study 3: Induction and recovery of dietary induced gastric ulcers inhorses4

Ulcers were induced in 12 Thoroughbred horses by simply using stable confinement, low level

exercise and high concentrate diet similar to that used in many Australian racing establishments over a

period of 10 weeks. Once ulcers were induced, the horses were turned out to pasture to investigate

how long it took them to recover from ulceration naturally, before an intervention experiment wascarried out (Study 4).

Protocol

There were 8 mares and 4 geldings, with a mean age of 7.7 3.5 years and an average weight of 478

37 kg.

1. Pre-trial pasture period

Horses had an initial 4 months pasture rest from August to November, representing predominantly

tropical spring pastures. Pasture size was approximately 10 ha and able to accommodate the horseswithout supplementary feeding. Pasture was typical SE QLD pasture with a predominance of kikuyu

and tropical grasses.

2. Induction period

This induction period was 10 weeks in duration and involved bringing the horses in and housing them

in a stable, feeding concentrate based diet and allowing exercise 5 days a week on a 12 bay horse

walker. Housing allowed horses to see and interact with other stabled horses. Exercise consisted of 10

minutes walking and 10 minutes trotting followed by 5 minutes walking to cool down. On days when

horses were not exercised on the walker they were turned out into a round yard during the cleaning of

their boxes in small groups of 2 to 4.

4 C.M. McGowan, T.W. McGowan, F.M. Andrews and R.A.M. Al Jassim. Induction and recovery of dietary

induced gastric ulcers in horses.Journal of Veterinary Internal Medicine, Volume: 21 Issue: 3 Pages: 603

Abstract: 115, 2007.

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Diet

All horses were fed the total diet as 2 equally divided meals twice daily

Acclimation period: Acclimation (2 wks, during weeks 0 -2)

Horses were fed lucerne (alfalfa) hay (6 kg) and oats (3 kg)

High roughage diet: (4 wks, during weeks 2 - 6)

Horses were fed lucerne hay (5 kg) + concentrate (4 kg)

Low roughage diet: (4 wks, during weeks 6 - 10)

lucerne hay (3 kg) + concentrate (5 kg)

Table 1. Chemical composition of the hay and concentrate mixture fed to horses

Chemical composition (%DM basis)

Feed DM% OM Ash NDF ADF CP DE MJ/kg DMHay 90.4 92.5 7.5 34.4 22.3 16.2 10.40

Concentrate 79.1 94.7 5.3 29.6 10.7 13.8 14.47

Legend: DM = dry matter, OM = organic matter, Ash = mineral ash, NDF = neutral detergent fibre,

ADF = Acid detergent fibre, CP = crude protein, DE = digestible energy

Table 2. Composition of the concentrate mix

Inclusion

Ingredients AS FED%

OATS (Whole or

Bruised) 35.70

BARLEY (Steam flaked) 30.00

CORN (Cracked) 14.00

Wheat Bran 4.00

Sunflower Seeds (Black) 4.00

Soyabean Meal 45.0 5.00

Molasses 5.00

Limestone 1.20

Di Calcium Phosphate 0.25

Salt 0.75

Dairy Vit/Min Premix 0.10

3. Post trial Recovery period

Following the endoscopy at week 10, horses were returned to pasture for a further 6 weeks (during

weeks 10 16).

This was during February to March and represented drier, Autumn pasture and so was supplemented

with lucerne (alfalfa) hay.

Endoscopy and stomach sampling

Gastroscopy examination using a 3 m Olympus endoscope was performed every 2 weeks from week 0.

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Horses were fed the evening before and had feed withheld the morning of the study. Water was

withheld during the final 2 hours before examination. Horses were restrained in breeding stocks and

sedated with xylazine and butorphanol to facilitate examination without use of other restraint

(Figure 1).

Figure 1. Gastroscopy examination procedure. From the left: Dr Rafat Al Jassim, Dr Thomas

McGowan, Horse 4, and Dr Catherine McGowan.

Complete gastroscopy examinations captured using computer digital video software and recorded.

Ulcers graded using the Number/Severity system (MacAllister et al., 1997) (Table 3) independently by2 people blinded to each other and the horse.

The average grade was used for analysis. As well as documenting the increase in ulcer lesions

endoscopically, small samples were taken throughout the study (every 2 weeks) of the gastric muscosa

and gastric contents and analysed as per study 1 for bacterial populations, investigating the

populations both in pastured horses, and those on high concentrate diets, and the bacterial involvement

in gastric ulceration.

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Table 3. Grading system for gastric ulcers based on MacAllister et al., 1997

Number Scores

0 = none seen

1 = 1-2 localised lesions

2 = 3-5 localised lesions

3 = 6-10 lesions

4 = > 10 lesions or diffuse (or very large lesions)

Severity Scores

0 = none seen

1 = Appears superficial (only mucosa missing)

2 = Deeper structures involved (greater depth than number 1)

3 = Multiple lesions and variable severity (1,2 and / or 4)

4 = same as 2 and has active = hyperaemic and/or darkened lesion crater

5 = same as 4 plus active haemorrhage or adherent blood clot

Study 4: Treatment of dietary induced gastric ulcers in horses

Involved re-induction of gastric ulceration using the method outlined above in study 3, yet once

ulceration was confirmed in 12 horses; they were divided into 3 groups. Group 0 horses were simply

control horses and were maintained on full rations with no treatment. Group 1 and 2 were also

maintained on full ration, but group 1 received a probiotic, a live bacterial culture of good bacteria,

based on the predominant lactic acid bacteria identified in study 1. The selected bacteria are known for

their probiotic characteristics and have the ability to adhere to stomach lining. Group 2 were given an

oral antibiotic. Samples as per study 1 for bacteriological analysis were also collected via endoscopy

and following the conclusion of this study, a representative number of horses from each group were

euthanased humanely and their stomachs harvested for full microbial analysis.

There were initially 15 Thoroughbred horses, 10 from the previous trial that had been rested at pasture

for 6 months, and 5 new horses that had an unknown period out of high intensity training. There were

8 mares and 7 geldings, with a mean age of 7.2 4.5 years and weighing an average of 485 39 kg.

Induction period

The induction period was 4 weeks in duration, following 1 week of acclimation where the concentrate

diet was introduced. Horses were housed in a stable, feeding concentrate based diet and allowing

exercise 5 days a week on a 12 bay horse walker. Exercise consisted of 10 minutes walking and 10

minutes trotting followed by 5 minutes walking to cool down. On days when horses were not

exercised on the walker they were turned out into a round yard during the cleaning of their boxes in

small groups of 2 to 4.

Diet

All horses were fed the total diet as 2 equally divided meals twice daily.

Acclimation period: Acclimation (1 wk, during weeks 0 -1)

Horses were fed lucerne (alfalfa) hay (5 kg) and concentrate mix (3 kg)

Induction diet: (8 wks, during weeks 1 - 9)

Horses were fed lucerne hay (3 kg) + concentrate mix (5 kg)

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Endoscopy and stomach sampling

Gastroscopy examination using a 3 m Olympus endoscope was performed in week 0, 3, 4, 7 and 9.

Horses were fed the evening before and had feed withheld the morning of the study. Water was

withheld during the final 2 hours before examination. Horses were restrained in breeding stocks and

sedated with xylazine and butorphanol to facilitate examination without use of other restraint(Figure 1).

Complete gastroscopy examinations captured using computer digital video software and recorded.

Ulcers graded using the Number/Severity system (MacAllister et al., 1997) independently by 2 people

blinded to each other and the horse.

The average grade was used for analysis. As well as documenting the increase in ulcer lesions

endoscopically, small samples were taken throughout the study (every 2 weeks) of the gastric muscosa

and gastric contents and analysed as per study 1 for bacterial populations, investigating the

populations both in pastured horses, and those on high concentrate diets, and the bacterial involvement

in gastric ulceration.

Treatments

At week 4, endoscopy revealed 3 horses that did not develop gastric ulceration and so they were

removed from the trial and the remaining 12 horses divided into 3 groups by RAJ so that neither of the

veterinarians performing the endoscopy knew which treatment group horses were in. There were 6

mares and 6 geldings, with a mean age of 7.3 3.8 years and weighing an average of 479 41 kg.

Treatment was started in week 5 and horses were examined after 2 and 4 weeks of treatment (weeks 7

and 9).

1. Probiotic preparation:

Lactobacillus agilis

Lactobacillus salivarius

Lactobacillus equi

Streptococcus equinus

Streptococcus bovis

Dose and concentration:

Final concentration was between 109

and 1010

viable cell per ml

Dose: 50 ml of the blend which provided 5 1010

to 5 1011

viable cells.

2. Oral antibiotic

Trimethoprim suphadimidine (Trimidine powder Parnell laboratories (Aust) pty ltd)

15 mg/kg bid per os.

3. Control

Horses continued with the diet, confinement and exercise as per study 1 for 4 weeks.

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Results

Study 1. The bacterial community of the horse stomach

The main findings of this study were 1.the diversity of bacteria adhering to the lining of the non-

glandular region of the stomach decreased during ulceration which could results in opportunisticpathogenic organisms to colonise the ulcers, 2. Cultured bacteria clustered with common bacterial

species belonging to the genera Lactobacillus, Streptococcus and Clostridium (Figure 2), while DGGE

clones were more diverse with main groups clustering with the genera Propionibacterium,

Clostridium, Lactobacillus, Prevotella, Pasteurella, , and Pseudomonas. Other fewer clones were

closely related toEscherichia,Actinobacillus,Moraxella,Rhodococcus, Veillonella, Legionella and

Eubacterium (Figure 3). Selected bacterial species on the basis of dominance and biochemical

characteristics were selected for use as probiotics in study 4.

1. Culture, isolation and identification of lactate-producing bacteria

The mean number of colony forming units in stomach contents cultured on modified MRS-agar

medium was 2.3 107. Forty-five colonies were picked and transferred to a broth of BM10 medium

supplemented with glucose (0.3%). Based on morphology, Gram stain reaction and RFLP profile,

sixteen isolates were identified by 16S rDNA sequencing. The sixteen isolates belonged to three main

groups closely related to the generaLactobacillus, Streptococcus and Clostridium (Figure 2). Two

isolates from ulcerated squamous tissue (UM21 and U31b) were closely related (98% identity) toLact.

agilis (M58803). Isolate G43, which was derived from the contents of healthy stomachs, was similar

(99%) toLact. equi (AB048833.1). Isolate G46 was closely related (99%) toLact. salivarius strain

RA2115 (AY389803.1, M59054) and isolate G1 was closely related (99.9%) to S. bovis (AF202263)

and S. equinus (X58318) (98.7%). Isolate G113 clustered with S. equinus (X58314). Isolates G31,

G34, G36, G315, G33, G310 and G32 originated from the stomach contents of healthy horses and

were all closely related ( 99%) to C. perfringens (AB045283). Isolate U31 from ulcerated stomach

contents clustered with C. butyricum (X68177) and G313 clustered with C. bifermentans (X75906).Similarities between the isolates and their closest known bacterial species were 90.8% and 84.9% for

U31 and G313, respectively.

Figure 2. Phylogenetic relationship of the derived sequences from 16S rDNA of cultured bacteria

2. Denaturing gradient gel electrophoresis profiles (DGGE)

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Initial analysis of DGGE profiles indicated apparent differences in bacterial diversity between sample

groups. Ulcerated tissues had less bacterial diversity than normal tissue from healthy stomachs. The

bacteria represented by the bands must have the capacity to adhere to the stomach lining because the

samples were washed with PBS.

Samples of stomach contents containing residual feed produced more bands than samples free of

residual feed, which indicates that the former samples had a more diverse bacterial community thanthe latter samples. The additional bands may represent bacteria associated with feed particles or

bacteria ingested with the feed. Sequences generated from DGGE clones comprised a short region

( 200 bp; V3) of the 16S rRNA gene and were not used to determine phylogenetic identity. However,

they were used to presumptively identify the different clones and relate them to database isolates.

Eighty percent of the 56 clones (25 DGGE bands) belonged to five main genera: Prevotella (29%),

Clostridium (14%), Pseudomonas (13%), Propionibacterium (13%) andLactobacillus (11%)

(Figure 5). Other clones (20%) clustered withEscherichia coli (n = 1),Legionella (n = 1), Voraxella

(n = 2) and Pasteurella (n = 4) (Figure 3).

Prevotell

ruminocola

29%

Clostridium spp

14%Lactobacillus spp

11%

Pseudomonas spp

13%

Voraxella canis

4%

Legionella

londiriensis

2%

Pasterell spp

7%

Eschirichia coli

2%

Rodococcus

rhodnii

5%Propionibacterium

spp

13%

Figure 3. Clones derived from denaturing gradient gel electrophoresis (DGGE) bands identified

using the V3 region of 16S rDNA. Genomic DNA was extracted from stomach contents and

stomach mucosa.

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Study 2: In vitro effects of hydrochloric and lactic acids onbioelectric properties of equine gastric squamous mucosa

Animals and gastric tissues

Thirteen horses aged from 2 to 33 years were recruited. 83% had low grade gastric ulcers in the

nonglandular mucosa. Tissue specimens were collected from grossly normal nonglandular mucosa at

or adjacent to the margo plicatus in the stomach of each horse, which is the region of the equine

stomach where most ulcers develop (Begg and OSullivan 2003). Sixteen tissue specimens were

collected from each of the 13 horses, making a total of 208 tissue specimens collected from this region.

The 13 tissues (1 from each horse), immediately placed in neutral-buffered 10% formalin, did not have

evidence of gross or histopathologic changes. Of the remaining 195 samples, 194 were analyzed in

Ussing chambers. One tissue sample was discarded due to a chamber malfunction. The variables

measured did not differ significantly between tissues from horses with ulcers and tissues from horses

without ulcers.

HCl exposure and recovered mucosa

Decreasing the pH from 7.0 to 1.5 and 4.0 caused Isc and PD to decrease significantly indicating

abnormal bioelectric properties. Mean Isc in tissues perfused with NRS at pH 7.0, 4.0 and 1.5

decreased 29%, 41%, and 41% from their initial values, respectively during the 270-minute exposure.

Mean PD across the tissues decreased by 50% in tissues exposed to NRS at a pH of 1.5, compared

with 5% from their initial values in tissues exposed to NRS at a pH of 7.0 and 4.0. Mean R or G did

not change over the exposure time.

After pH was adjusted to 7.0, mean Isc and PD immediately increased to near control values in tissues

exposed to NRS at a pH of 1.5. Analysis of these data suggests that sodium transport in the

nonglandular tissue and barrier function recovered once pH was increased to 7.0. Thus, the affect of

HCl on sodium transport and barrier function is reversible; however prolonged exposure may lead to

irreversible tissue damage and ulceration.

Lactic acid exposure and recovered mucosa

Mean Isc in tissues perfused with all concentrations of LRS at a pH of 4.0 and 7.0 did not significantly

change from control tissues. Although there were no significant changes in tissue parameters during

the study, there were trends toward and increase in tissue conductance and decrease in tissue

resistance. It may be that LA requires a longer exposure than the 270 minutes to cause significant

damage or may act synergistically with VFAs to cause significant acid injury.

Mannitol fluxes

Because tissue exposed to a 40 mM concentration of LRS showed a trend toward increased G and

lower R, nonglandular tissues (n=2) were exposed to LRS (40 mM) in [14C] mannitol at pH 1.5. These

tissues showed increased permeability to [14C] mannitol when compared to tissues exposed to the

same level of LA at pHs 4.0 and 7.0. The increase in cell permeability and conductance with a

corresponding decrease in tissue R may indicate that higher concentrations of LA at a low pH may

cause damage to the nonglandular mucosa by disrupting paracellular spaces and allow leakage of acid

between and into cells causing ulcers.

LA and mucosal histopathologic changes

Histologic examination of specimens of nonglandular mucosa exposed to NRS and LRS, at various

concentrations, at pH of 4.0 and 7.0 were normal. On the other hand, mucosa exposed to NRS at a pHof 1.5 had multifocal cellular swelling and a mottled appearance in the superficial stratum corneum

and stratum transitionale. Mucosa exposed to LRS at the various concentrations and pHs did not show

cell swelling in the stratum transitionale and stratum spinosum other than was observed in tissues

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exposed to NRS alone at the same pH. Thus, exposure of nonglandular mucosa to LRS (5, 10, 20, or

40 mM) a low pH did not result cell swelling consistent with damage to cellular sodium transport. The

increased nonglandular mucosa permeability caused by LRS (40 mM) may be related to extracellular

mechanisms.

Study 3: Induction and recovery of dietary induced gastric ulcers in

horses

During the induction period 11 of 12 horses developed ulcers. The one horse that did not develop

ulcers had ulcers at beginning and again 4 weeks on pasture, but not during the treatment period. Only

one horse with ulcers (6) showed overt clinical signs (refusal of concentrate, and weight loss) but four

others chose to eat roughage prior to concentrate. Also, after initial increase, there was a reduction in

bodyweight on the high carbohydrate and restricted roughage diet, in weeks 6 and 8 (P


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0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 12 14 16

Week

Number Score

Severity Score

Figure 5. Mean gastric ulcer score for 13 horses fed concentrate ration and being confined

during weeks 0 10 and subsequently during pasture recovery during weeks 10-16. Number and

severity are shown separately as blue and purple respectively

The mean ulcer score of horses of horses in weeks 8 and 10 was approximately 3 (Figure 6, Table 3)

demonstrating the severity of ulceration that would be common among similarly managed horses in

the equine industry.

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Figure 6. Endoscopic images of severity grade 3 ulceration in two horses

There were, however, a few surprising findings. Although they were mostly low grade, ulcers were

found in 5 horses after 4 months pasture at the beginning the study (week 0). Severity: mean 0.54

0.78; Number: mean 1.33 1.74.

Further, ulcer lesions were not resolved in 45% horses and new lesions occurred in 42% horses during

6 wks pasture recovery period. New lesions were diffuse superficial lesions but had a lot of

inflammatory reaction associated with them.

These results clearly demonstrate that confinement and feeding of high concentrate diets is sufficient

to induce gastric ulceration in horses, without intense exercise. Gastric ulcers may not heal on pasturein a 6 week period in a proportion of horses and further that new, albeit low grade, ulcers can develop

in pastured horses.

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Study 4: Treatment of dietary induced gastric ulcers in horses

During the induction period of Study 4, mean ulcer score increased similarly to study 3, to a peak

score of 3.420.79 (number) and 2.42 0.79 (severity) in week 4, following 4 weeks of high

concentrate diet (Figures 7 (number score) and 8 (severity score)).

During the treatment period, mean ulcer grade decreased from week 4 to week 9 overall to a mean of2.001.6 (number) and 1.421.24 (severity).

When groups were examined separately, ulcer scores were not significantly different at week 4. Means

for groups 0 (control), 1 (Probiotic) and 2 (TMPS) were 3.750.50 (number) and 2.75 0.50

(severity); 3.250.96 (number) and 2.25 0.96 (severity); and 3.250.96 (number) and 2.25 0.96

(severity) respectively.

However in week 9 means for groups 0 (control), 1 (Probiotic) and 2 (TMPS) were 3.250.5 (number)

and 2.25 0.96 (severity); 2.251.50 (number) and 1.50 1.29 (severity); and 0.501.00 (number) and

0.50 1.00 (severity) respectively.

Note that ulcers continued to increase in severity in comparative weeks (5-9) on the same diet and

exercise regimen as study 3. This shows that oral antibiotics (TMPS) and to a lesser extent oral

administration of probiotics reduce ulcer lesion number and severity compared to controls horses in a

4 week period.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Trial 2

WK_0

number

Trial 2

WK_3

number

Trial 2

WK_4

number

Trial 2

WK_7

number

Trial 2

WK_9

number

Control Group

Pro-biotic Group

Antibiotic Group

Figure 7. Graph representing the mean number scores for the 3 different groups in the study(Control, Pro-biotic, and Antibiotic) for each week during trial 2 (95% CI error bars)

18


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0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Trial 2

WK_0

severity

Trial 2

WK_3

severity

Trial 2

WK_4

severity

Trial 2

WK_7

severity

Trial 2

WK_9

severity

Control Group

Pro-biotic Group

Antibiotic Group

Figure 8. Graph representing the mean severity score for each of the 3 groups (Control,Probiotic and Antibiotic) for each week during the study

Analysis of the Severity of gastric ulceration

ANOVA was used to assess for differences in the mean severity scores between weeks 0 and 4 within

each group and between weeks 4 and 9 within each group. The Control Group and the Pro-biotic

Group mean severity scores were significantly higher in week 4 as compared to week 0 (p=0.003 and

p=0.042, respectively), but not significantly different from week 4 to week 9. The antibiotic group

mean severity score was significantly higher in week 4 as compared to week 0 (p=0.013) and week 9

was significantly lower than week 4 (p=0.027). There was no significant difference in mean scores of

severity between week 3 and week 4 for any of the 3 groups.

There was no significant difference of the mean severity of ulcer scores among the 3 groups in weeks

0, 3, or 4. There was a trend (p=0.067) for the antibiotic group to have a lower mean score of severity

of gastric ulcers as compared to the control group in week 7. There was no significant difference

between the mean severity scores of the Control Group compared to the Pro-biotic Group or between

the Pro-biotic group and the antibiotic Group in week 7. The mean of the severity score of the Control

Group was significantly higher than that of the antibiotic group (p=0.05) when analysis of the means

of severity scores was performed across the 3 groups within week 9. There was no significant

difference between the mean severity score of the Control Group and the probiotic Group, nor was

there a significant difference in the mean severity scores between the Pro-biotic group and the

Antibiotic Group.

Analysis of the Number of gastric ulceration

There was no significant difference in the means of numbers of gastric ulcers among the 3 groups

(Control Group, Pro-biotic Group and antibiotic Group) when compared across the weeks 0, 3, and 4.

There was a trend for the mean score of the antibiotic group to be lower than that of the Control Group

(p=0.072). There were no other significant differences in week 7. In week 9, the mean score for the

number of ulcers in the antibiotic Group was significantly lower than the mean score for the Control

Group (p=0.006).

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Discussion of results

This study has achieved its aims by increasing the knowledge of gastric ulceration in horses, and

highlighting how bacteria play a role by contributing to increased acidity of the stomach when horses

fed concentrate diet, by identification of the different bacterial groups in ulcerated and healthy

stomachs, by identification of bacteria in ulcers in vivo and by the response of horses to antibiotic andprobiotics in markedly reducing gastric ulceration under proven ulcerogenic conditions.

Bacterial Community of the equine stomach

The results of study 1 have confirmed the diverse number of bacterial species that live in the equine

stomach, many of which are acid tolerant and capable of fermenting starch and other readily

fermentable carbohydrates and produce lactic acid which is along with the common short chain fatty

acids produced could have a role in damaging the mucosal lining of the stomach.

The culture-dependent technique provided evidence of the selectivity of the stomach environment for

acid-tolerant bacteria of two main genera:Lactobacillus and Streptococcus, both are lactate producers.

Other culturable bacteria were mainly spore-forming pathogenic clostridia, including C. perfringens

and C. botulinum. These pathogenic bacteria are known as low-GC Gram positive and endospore

forming bacteria that produce toxins responsible for gastroenteritis in humans and animals. The source

of these clostridia was probably the collection yard because soil is their main habitat, where they live

primarily in pockets rendered anoxic by other facultative organisms that metabolize organic

compounds. Horses ingest a range of environmental microorganisms while searching for feed in the

yards where they are held before slaughter. The presence of these bacteria in the stomach may be a

temporary phenomenon and cause no harm to the horse when the microbial population of the

gastrointestinal tract is balanced and the stomach and intestinal lining is healthy and colonised by the

friendly bacteria. However, it is not known whether they have a functional role in the stomach or

whether they produce toxins while resident in the stomach. The presence of high numbers of bacteriain the stomachs of fasted horses (2.3 107) has not been documented. The bacterial count in the

stomach is generally expected to be very low because of its acidity, which acts as a chemical barrier to

entry of microorganisms to the intestine. However, it is important to note that a pH gradient exists in

the stomach of the horse; the non-glandular region is less acidic than the rest of the stomach and

enables acid-tolerant bacteria to grow.

The molecular techniques used in this study overcame the problem of media selectivity and revealed a

diverse bacterial community. These techniques were based on analysis of genomic DNA, which was

derived from all bacteria present in the sample regardless of whether they were an established

community or were transient. In addition to the main bacterial groups that were identified by culture

on MRS agar medium, bacteria belonging to the genera Pseudomonas, Prevotella and

Propionibacterium were identified using DGGE analysis. Other genera identified wereEscherichia,Legionella, Voraxella and Pasteurella. Although these were less prevalent than the other genera and

were derived from fewer clones, they may play a role in causing disease under certain conditions.

Further work is required to determine the roles of the various groups of bacteria, particularly the

culturable species, in the pathogenesis of stomach ulceration in horses. Research with rats suggests

that stomach bacteria may colonize gastric ulcers caused by acid damage and delay healing (Elliott

et al., 1998).

Work underway to complete the analysis of DNA samples that has been obtained from ulcers and

healthy stomachs of horses used in ulcers induction trials. Preliminary data indicates that good bacteria

may lose their ability to colonise the stomach lining when ulcerated. Most biopsies from ulcerated sites

produced poor DNA after washing with saline solution compared with that from healthy region.

The role of lactic acid

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These results indicate that exposure of the non-glandular squamous mucosa of the stomach to lactic

acid in the presence of HCl results in reduction of mucosal barrier integrity, which enables HCl to

diffuse into tissue layers immediately deep to the stratum corneum of the non-glandular mucosa. This

may undermine the superficial mucosa and cause ulceration. Although the damage observed in these in

vitro experiments was mild compared to those observed after exposure to high concentrations of

VFAs, a longer exposure may be needed to induce a similar extent of acid injury. Some horses may be

more susceptible to the damaging effects of lactic acid than others, which would explain why somehorses develop ulcers and others do not. The presence in gastric fluid of lactic acid, a by-product of

bacterial fermentation, may contribute to gastric ulcer disease and may partly explain why diets

high in soluble carbohydrates have been associated with the development of gastric ulcers. Further in

vivo research is needed to determine the effects of lactic acid alone and in combination with other by-

products of fermentation such as VFAs to ascertain whether these compounds exhibit synergy in

altering mucosal bioelectric properties.

Induction of gastric ulcers

Ulcers occurred in stabled horses on high concentrate, low roughage diet without intense exercise

stimulus. It is not known how much of the stimulus could be related to housing as housing alone has

been shown to have a role in the development of gastric ulceration in horses (Murray and Eichorn

1996). However, the increasing severity with increased concentrate and decreased roughage supports

the contention that the diet had a large role in the development of the widespread severe ulceration in

horses in this trial.

The other interesting finding in this study was to observe the lesions over time. It appears that severity

score 1 lesions also represent early lesions which contract and form a crater (severity 2) lesions over a

number of days. Some of these ulcers will resolve while in other situations, especially in intensively

managed horses, the ulcers progress to deeper lesions and do not resolve.

In our research, we noted that ulcers did not resolve on pasture and further, that new ulcers developed.

This is supported by recent research in New Zealand in racehorses trained from pasture (Bell et al.,2007). It seems that horses on pasture do have ulceration, but these tend to cycle through grade 1 to 2

severity and not progress. The results of this study support the hypothesis that the non-resolving

lesions represent lesions colonised by bacteria where bacterial products further damage and allow

progression rather than healing of gastric ulceration.

Treatment of gastric ulcers

Further supporting our original hypothesis of the involvement of bacteria in the progression and

severity of gastric ulceration are the results of this final study. Despite horses being managed exactly

the same way as in Study 3 where ulceration continued to increase in severity across the group, horses

being treated with an oral antibiotic had a significant and dramatic reduction in their gastric ulcer

severity score. The reduction was evident as a trend in just two weeks, but was fully significant as 4weeks. These results are as good as or better than the results from antacid treatments.

However, even more importantly was the indication that probiotics may do the same. The small

volume of live bacteria given orally per day was able to reduce the severity of ulcers even in this small

group. While these results were not statistically significant the trend is there, and in a small study of

just four horses, is enough to warrant further research to test the possibility of a response in a larger

number of horses in the natural situation. Use of a probiotic instead of an antibiotic has huge

advantages in avoiding use of antiobiotics that can promote bacterial resistance, and avoiding

expensive and potentially controlled medications for competition.

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Implications

The implications of this research are that we have established the role of bacteria in the pathogenesis

of gastric ulceration in horses through laboratory experiments and studying gastric ulceration in the

live horse. There is a great opportunity to further investigate the use of antibiotics/and or probiotics for

treatment of ulcers in horses. A role for modification of microbial populations in the future treatmentof gastric ulceration has been shown and the future use of probiotics and other non-medical

manipulations of microbial populations in horse predisposed to gastric ulceration is a promising

prospect.

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Recommendations

We recommend further studies on the use of different live bacterial probiotics on the prevalence of

gastric ulceration. We also recommend that restriction of roughage in horses on a high grain diet

should be avoided and that horse owners feeding their horses five kg high grain based concentrate diet

per day should be aware of the very high prevalence of gastric ulceration under this regimen,especially if the horse is concurrently confined to a stable.

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Appendices

List of presentations

R. Al Jassim, An abstract/poster was presented at the RRI-INRA gut microbiology meeting in

Aberdeen, Scotland 21-23 June 2006.

R. Al Jassim, Seminar was presented at the College of Veterinary Medicine, University of Tennessee

June 30, 2006.

R. Al Jassim, Plenary talk at the Recent Advances in Animal Nutrition in Australia meeting, Armidale,

Australia, July 9-11, 2007.

R. Al Jassim, Plenary talk at The VII International Symposium on the Nutrition of the Herbivores,

Beij

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Gastric Ulceration in Horses the Role of Bacteria and Lactic Acid

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